Real-time mass spectrometry in enzymology

Abstract

IntroductionResearch on enzymatic (catalysis) reactions is of largeinterest since it is known that many proteins have abiological function. Thus the word “enzymology” encom-passes the branch of biochemistry dealing with the chemicalnature and biological activity of enzymes [1], and thusresearch on their function and activity, and on inhibition oftheir reactions. In recent times the word has been used in avariety of other research fields, for example clinical(medical) enzymology, cryoenzymology, engineering enzy-mology, micellar enzymology, metal enzymology, soil en-zymology, and others [2]. Since “functional proteomics”became a very productive research field [3–5], enzymologyhas found new popularity, especially due to advances inmass spectrometric detection.In the last twenty years atmospheric pressure ionization–mass spectrometry (API–MS) has achieved acceptance as animportant analytical tool in biochemical research. Theeffective coupling of liquid chromatography with MS laidthefoundationforasensitive,specific,andspeedytechniquestudyingmoleculesfromcomplexbiologicalsources.Thus,inthe last five years over a dozen ABC Trend articles haveappeared on mass spectrometry, including technical develop-ments and biological approaches [6–18]. In summary, all ofthese predict MS has a successful future as a detector.The current state of enzymatic real-time MSGenerally, functions of proteins, their activity and regula-tion can be studied in vivo and in vitro. In vitro assaysrequire enzymes which are active in an artificial environ-ment and which can be isolated, expressed, or synthesizedin manageable amounts. Although in vitro assays often lackphysiological conditions, the results obtained give rudi-mentary information about proteins and their catalyticpotential. Furthermore, in contrast with in vivo assays,single enzymes can be handled and regulated more easily.A typical enzymatic in vitro assay consists of:1. starting the reaction by mixing the reaction compounds;2. taking aliquots after different reaction times;3. interrupting the reaction in each aliquot;4. separating the reaction compounds; and5. detecting the compounds for quantification.Classical methods use spectroscopic and/or radioisotopicdetection for step 5. However, disadvantages of bothtechniques are the need for substance labeling and, after-wards, very difficult identity differentiation between theindividual non-labeled reaction components. With massspectrometry (API–MS) the labels become redundant andseveral compound ions can be identified. This also enablesresearch on enzymatic reactions involving various types ofmolecule (e.g. hydrophobic substrates, semi-polar inter-mediates, and/or polar products).From the very first day, API–MS was applied in hyphen-ation with liquid chromatography (LC) for compound quanti-ficationandidentification.Gener ally,thisprinciplewasutilizedon the basis of LC–UV–visible and LC–fluorescence. Todaythis method is mainly used for protein identification viapeptides obtained from proteases like trypsin [4, 5] but, never-